Since the development of plant transformation systems a particular interest exists on the stable expression of foreign genes in higher plants. Although these attempts were successfully realized for several herbicide resistance genes, a series of experiments failed that had been designed to use plants as efficient bio-reactors. It seemed that higher plants have a system of defense to protect themselves against overexpression of foreign genes. Whenever a transgene expression comes up to a threshold dose it is without selective pressure silenced either by transcriptional or post-transcriptional inactivation. Recent studies on these phenomena indicated that “antisense” RNA might be responsible for these silencing mechanisms. The idea of an “antisense” RNA-mediated gene silencing was substantiated by the analysis of transgenically mediated virus resistance in plants (Smith et al., 1994; English et al., 1996; Sijen et al., 1996). It could be shown that the in vivo transcripts of non-translatable “sense” cDNA constructs which had been integrated into the plant genome can mediate resistance against Infection with plant viruses. Further analysis revealed that this resistance which was due to post-transcriptional gene silencing (PTGS) only occurred when the transgene shared homology with the Infecting virus. Based on these data and with regard to previous results describing the co-suppression phenomena in plants (for review see Meyer, 1996) several models explaining the PTGS mechanism have been introduced (English et al., 1996; Baulcomb, 1996; Sijen et al., 1996). Common to all these models is the assumption that “antisense” RNAs are synthesized from “sense” RNA templates by an RNA-directed RNA polymerase (RdRP). Subsequently the produced “antisense” RNAs can hybridize to complementary parts of mRNAs or invading virus RNAs which would unresistingly lead to degradation of the double-stranded regions.
It is most likely that this PTGS is not restricted to transgenes but that it is also involved in normal plant gene regulation. Moreover, it is most likely that the same mechanism takes place in all higher eukaryots. Therefore, a detailed examination of RNA-mediated gene regulation had become an important aspect of basic and applied research. An RNA-dependent RNA polymerase (EC 2.7.7.48, RdRP) activity has been detected in healthy plant tissue, for review see Fraenkel-Conrat (1986). Previous studies of RdRP activity have suffered from the inhomogeneity of enzyme preparations and of the resulting RNA products in that they did not allow the precise determination of their template complementarity by direct RNA sequencing. Schiebel et al. (1993 a, b) provided evidence for the concept that the RdRP mediated transcription is a truly RNA-instructed process yielding products that are precise complementary copies of the RNA template offered to an RdRP active enzyme preparation. However, although purification of the RdRP from tomato leaf tissue to electrophoretic homogeneity was reported (Schiebel et al., 1993 a, b), the enzyme preparations were not approachable for amino acid sequencing. One reason for the slow progress in elucidating the amino acid sequence of the RdRP is to be seen in the fact that only low amounts of protein could be isolated using the hitherto available technology and/or that there existed further proteins in the enzyme preparations obtainable with standard techniques which resulted in non-informative or even false sequence information. However, for efficient use of RdRP associated technology, it was desirable to be able to manipulate the genetic material associated with said technology.
Thus, the technical problem underlying the present invention is to provide such material.